Design, Synthesis, and Antiviral Activities of New Benzotriazole-Based Derivatives

Several human diseases are caused by enteroviruses and are currently clinically untreatable, pushing the research to identify new antivirals. A notable number of benzo[d][1,2,3]triazol-1(2)-yl derivatives were designed, synthesized, and in vitro evaluated for cytotoxicity and antiviral activity against a wide spectrum of RNA positive- and negative-sense viruses. Five of them (11b, 18e, 41a, 43a, 99b) emerged for their selective antiviral activity against Coxsackievirus B5, a human enteroviruses member among the Picornaviridae family. The EC50 values ranged between 6 and 18.5 μM. Among all derivatives, compounds 18e and 43a were interestingly active against CVB5 and were selected to better define the safety profile on cell monolayers by transepithelial resistance test (TEER). Results indicated compound 18e as the hit compound to investigate the potential mechanism of action by apoptosis assay, virucidal activity test, and the time of addition assay. CVB5 is known to be cytotoxic by inducing apoptosis in infected cells; in this study, compound 18e was proved to protect cells from viral infection. Notably, cells were mostly protected when pre-treated with derivative 18e, which had, however, no virucidal activity. From the performed biological assays, compound 18e turned out to be non-cytotoxic as well as cell protective against CVB5 infection, with a mechanism of action ascribable to an interaction on the early phase of infection, by hijacking the viral attachment process.


Introduction
For centuries, infectious diseases have been the leading cause of death. Unfortunately, this sad record is still upheld today, primarily in the poorest countries; however, it was sadly proved that the pandemic risk was not negligible even in the most advanced countries and viral diseases must be considered a threat that we should not underestimate.
Within the past century, multiple viruses caused pandemics across the word. It is worth mentioning the Spanish flu , which was due to the H1N1 virus, which caused 50 million deaths [1], the Asian flu (1957), caused by the H2N2 virus [2,3], and the Hong Kong avian flu (1968), which originated from the H3N2 virus [4]. In recent history, HIV infection, causing AIDS, was arguably the most important huge viral infection, that claimed more than 36 million deaths, according to the World Health Organization (WHO) [5]. In the twenty-first century, SARS (severe acute respiratory syndrome), an atypical and particularly severe form of pneumonia, appeared in 2003 across a Chinese region and registered several cases in eight months, with a lethality rate of 10%; it was caused by SARS-CoV [6]. Later, in 2009, swine flu occurred across the American continent, caused by a H1N1 strain [7]. In 2012, another coronavirus, MERS-CoV, was identified as the cause of Middle East respiratory syndrome (MERS) [8]. Two years later, the Ebola virus was responsible for a new epidemic wave with a death rate of 50-90% [9]. Lastly, in 2019, SARS-CoV-2 circulated across the world causing the current COVID-19 pandemic [10], the effects of which we still experience.
Even though the World Health Organization (WHO) undertook specific control and research programs for the most known viral infections (AIDS, hepatitis C, avian flu and Dengue fever) and their impact in terms of morbidity and mortality was significantly reduced compared to the past, they still remain important global public health challenges [11]. Numerous other infections caused by emerging viruses (Chikungunya, Zika) are considered very threatening by the WHO and, therefore, some of them were placed under observation, while some others were indicated as future risks for epidemics.
Unfortunately, for most human pathogenic viruses, no vaccines or specific drugs are available, and the existing treatments are essentially symptomatic. In a few cases, targeted therapies are available (e.g., HIV, HCV), allowing access to acute and chronic treatments.
Among the currently untreatable viruses, the enteroviruses family includes seven species: human enterovirus A-D (referred to as poliovirus, Coxsackievirus A, Coxsackievirus B, echovirus and enterovirus in the previous taxonomy) and rhinovirus A-C. These viruses can trigger numerous diseases such as common cold, non-differentiated febrile diseases, hand, foot, and mouth syndrome, epidemic pleurodynia, herpangina, poliomyelitis, aseptic meningitis, myocarditis, and various respiratory infections [12]. A benign prognosis was observed in most cases, but severe respiratory disturbances [12] or other serious complications, such as dilated cardiomyopathy or flaccid myelitis frequently occur in infants or immuno-compromised adults [13][14][15]. Due to their high diffusion in many states [16][17][18], surveillance programs for diseases caused by enteroviruses are active, confirming their significant incidence in both pediatric and adult populations. Recently, many studies also reported a peculiar correlation between enterovirus infections and celiac disease or diabetes [19,20], and therefore, they undergo specific surveillance [21][22][23]. With the worrying spread of enterovirus infections and the lack of targeted therapies and preventive vaccines, except for poliovirus and two inactivated enterovirus A71 only approved in China for the prevention of hand, foot, and mouth human disease [24], the search for therapeutic agents able to tackle or heal enteroviruses-caused infections is of great relevance and usefulness. Although, in recent years, many synthetic compounds showed good antiviral activity against enteroviruses, they failed to enter the clinic. For instance, pleconaril was rejected by the FDA due to a set of adverse effects, while vapendavir failed a phase IIb trial due to insufficient efficacy [25][26][27]. Hence, basic antiviral drug discovery programs still represent a valid contribution to the development of new antiviral compounds. Under such circumstances, the present study investigated the antiviral activities of newly synthesized benzotriazole derivatives on a panel of selected viruses.

Rationale
Nitrogen-based heterocycles represent a precious source of scaffolds for the development of therapeutic agents in medicinal chemistry. Most of the FDA-approved drugs possess the N-heterocycle skeleton [28], which can be differently functionalized to obtain different compounds endowed with biological properties and, therefore, various pharmacological applications [29]. A wide number of nitrogen-containing heterocyclic derivatives were recognized to exhibit a broad range of pharmacological effects [30][31][32][33][34] and were widely reported in the literature to show intriguing biological activities for these types of compounds [35][36][37].
obtain different compounds endowed with biological properties and, therefore, various pharmacological applications [29]. A wide number of nitrogen-containing heterocyclic derivatives were recognized to exhibit a broad range of pharmacological effects [30][31][32][33][34] and were widely reported in the literature to show intriguing biological activities for these types of compounds [35][36][37].
Aiming to expand the structure-analysis knowledge on these types of molecules, in this work, we present new derivatives obtained through the manipulation of our previously developed hit compounds. Notably, we previously observed that the introduction of a methylene spacer on the not active N1,N4-bis(4- (5,6-dichloro-1H-benzo[d] [1,2,3]triazol-1-yl)phenyl)succinimide derivative (A, Figure 3) successfully led to N1,N4-bis(4-((5,6dichloro-1H-benzo[d] [1,2,3]triazol-1-yl)methyl)phenyl)succinimide derivative (B, Figure  3), which was active on CVB at micromolar concentration (EC50 = 23 μM) [43].  [1,2,3]triazol-1-yl)phenyl)succinamide (A) and its active derivative N1,N4-bis(4-((5,6-dichloro-1H-benzo[d] [1,2,3]triazol-1-yl)methyl)phenyl)succinamide (B). Therefore, the structural modifications were mainly focused on a) the introduction of a methylene spacer between the benzotriazole moiety and the para-substituted benzene ring and b) the position variation of the substitution on the aromatic ring, as exemplified in Figure 4. The structural manipulations were rationally designed to allow a greater degree of molecule flexibility to evaluate the effects induced by different molecular conformations. Therefore, the structural modifications were mainly focused on a) the introduction of a methylene spacer between the benzotriazole moiety and the para-substituted benzene ring and b) the position variation of the substitution on the aromatic ring, as exemplified in Figure 4. The structural manipulations were rationally designed to allow a greater degree of molecule flexibility to evaluate the effects induced by different molecular conformations.

Chemistry
To obtain the desired compounds, firstly, the amine intermediates were synthe via the two-steps synthetic routes reported in Scheme 1, which yielded the uniquely line derivatives 5b, 6a,b, 11b, 12a-e. The appropriate benzotriazoles 1a-e were conde with 3-or 4-nitrochlorobenzyl (2, 7, respectively) in a basic environment for Cs2CO3 obtained products 3a,b, 4a,b, 8a-e, 9a-e, and 10e were always obtained as a mixtu isomers, which were separated by flash chromatography. Each of them was subject

Chemistry
To obtain the desired compounds, firstly, the amine intermediates were synthesized via the two-steps synthetic routes reported in Scheme 1, which yielded the uniquely aniline derivatives 5b, 6a,b, 11b, 12a-e. The appropriate benzotriazoles 1a-e were condensed with 3-or 4-nitrochlorobenzyl (2, 7, respectively) in a basic environment for Cs 2 CO 3 . The obtained products 3a,b, 4a,b, 8a-e, 9a-e, and 10e were always obtained as a mixture of isomers, which were separated by flash chromatography. Each of them was subjected to a reduction reaction with methylhydrazine in autoclave in the case of chlorinated derivatives 4a, 8a, 9a, while in all other cases, hydrated hydrazine with palladium on activated charcoal was used, refluxed in ethanol. Not all reductions were successful; the gained amines (5b, 6a,b, 11a,b,d, 12a-e) were obtained in fair to good yields. Final derivatives 23b-34b, 75b, and 76b were synthesized as reported in Scheme 2, while the remaining are presented in Scheme 3.
The proper anhydride I (acetic anhydride, propionic anhydride, butyric anhydride and pivalic anhydride) at room temperature or for 1-72 h. The crude products were in turn obtained pure or required purification by flash chromatography; ii. The required benzoyl chloride derivatives II in N,N-dimethylacetamide (DMA) or N,N-dimethylformamide (DMF) at 80 °C from 3 h to 7 days. The purification of the compounds was carried out by recrystallization from ethanol or by flash chromatography; Final derivatives 23b-34b, 75b, and 76b were synthesized as reported in Scheme 2, while the remaining are presented in Scheme 3.
Among the whole series of synthesized compounds, only about 15% of which showed antiviral activity, and they were the sole reported in Table 1. The non-active compounds were withheld to simplify the table readability. Most of the appealing compounds (6a, 11b, 11d, 18e, 25b, 41a, 43a, 99b, 100b) were found selectively active against CVB5 with EC 50 values ranging between 6 and 52 µM, when non-inhibitory activity against the remaining virus replication was detected. Non-substituted benzotriazole-based compound 86c was selectively active against BVDV (EC 50 = 3 µM), while compound 21e preferentially inhibited RSV (EC 50 = 20 µM).
Overall, the most promising and effective derivatives are 18e and 43a, whose EC 50 values were 12.4 and 9 µM, respectively, when tested against CVB5.  To further outline the structure-activity relationships (SARs), Figure 5 reported the structures of the most active compounds together with precursors and the corresponding EC 50 values. Favorable chemical manipulations are indicated with a blue arrow, while unfavorable modifications are indicated with a red one. The intermediate 6a, bearing the side chain on position C-3′, presented moderate activity towards CVB5, with an EC50 of 52 μM. Notably, the substitution of the amine group with 3,4,5-trimethoxybenzoyl or p-chlorobenzoyl groups increased the antiviral activity against the same virus, with EC50 values decreased to 18.5 and 9 μM for compounds 41a and 43a, respectively. The latter compounds bore two chlorine atoms on C-4 and C-5 of the benzotriazole scaffold (in Figure 5A). The chlorine atoms seemed to be responsible for the greater activity, since their replacement with methyl groups led to inactive derivatives 51b and 53b (data not shown). Aliphatic amides (35a and 36a) and urea derivatives (58a-62a) obtained from chlorinated intermediate 6a showed no antiviral activity (data not shown). Aromatic amide moiety is allegedly required for anti-CVB5 activity. Concerning the C-4′-aminobenzyl derivatives, dimethyl benzotriazole-based aliphatic-urea compounds 99b and 100b showed a moderate anti-CVB5 activity resulting in EC50 values of 16 and 50 μM, while corresponding aliphatic amides 77b and 78b were revealed to be inactive (in Figure 5B, data not shown). When aliphatic-urea steric hindrance was increased from 99b to 100b, activity decreased.
A remarkable SAR analysis may be described when compound 21e is compared with 18e derivative. The former was active against RSV, while the latter inhibited the CVB5 viral replication. The two derivatives both shared the 4-F benzotriazole intermediate 12e, but derivative 21e carried on a trimethoxy-phenyl amide moiety, while 18e was the simplest pivalamide (in Figure 5C).
Among all derivatives, we selected compounds 18e and 43a for their interesting activity against CVB5, with comparable CC50 and EC50 values, and further experiment were The intermediate 6a, bearing the side chain on position C-3 , presented moderate activity towards CVB5, with an EC 50 of 52 µM. Notably, the substitution of the amine group with 3,4,5-trimethoxybenzoyl or p-chlorobenzoyl groups increased the antiviral activity against the same virus, with EC 50 values decreased to 18.5 and 9 µM for compounds 41a and 43a, respectively. The latter compounds bore two chlorine atoms on C-4 and C-5 of the benzotriazole scaffold (in Figure 5A). The chlorine atoms seemed to be responsible for the greater activity, since their replacement with methyl groups led to inactive derivatives 51b and 53b (data not shown). Aliphatic amides (35a and 36a) and urea derivatives (58a-62a) obtained from chlorinated intermediate 6a showed no antiviral activity (data not shown). Aromatic amide moiety is allegedly required for anti-CVB5 activity. Concerning the C-4 -aminobenzyl derivatives, dimethyl benzotriazole-based aliphatic-urea compounds 99b and 100b showed a moderate anti-CVB5 activity resulting in EC 50 values of 16 and 50 µM, while corresponding aliphatic amides 77b and 78b were revealed to be inactive (in Figure 5B, data not shown). When aliphatic-urea steric hindrance was increased from 99b to 100b, activity decreased.
A remarkable SAR analysis may be described when compound 21e is compared with 18e derivative. The former was active against RSV, while the latter inhibited the CVB5 viral replication. The two derivatives both shared the 4-F benzotriazole intermediate 12e, but derivative 21e carried on a trimethoxy-phenyl amide moiety, while 18e was the simplest pivalamide (in Figure 5C).
Among all derivatives, we selected compounds 18e and 43a for their interesting activity against CVB5, with comparable CC 50 and EC 50 values, and further experiment were performed to better define the safety profile on cell monolayers by transepithelial resistance test and deeply analyze the mechanism of anti-CVB5 action.

Transepithelial-Transendothelial Electrical Resistance (TEER) Test
In parallel with the low cytotoxic profile shown by our derivatives against evaluated cell lines, compounds 18e and 43a were selected to ascertain their potential toxic effect on human cells. We tested this on differentiated intestinal Caco-2 monolayers, commonly used to simulate the gut epithelium and to evaluate changes in intestinal permeability. Cells were treated with the bacterial endotoxin lipopolysaccharide (LPS) as a negative control, and it was observed that it caused permeability imbalance and a significant alteration of the cell monolayer integrity with time ( Figure 6

Transepithelial-Transendothelial Electrical Resistance (TEER) Test
In parallel with the low cytotoxic profile shown by our derivatives against evaluated cell lines, compounds 18e and 43a were selected to ascertain their potential toxic effect on human cells. We tested this on differentiated intestinal Caco-2 monolayers, commonly used to simulate the gut epithelium and to evaluate changes in intestinal permeability. Cells were treated with the bacterial endotoxin lipopolysaccharide (LPS) as a negative control, and it was observed that it caused permeability imbalance and a significant alteration of the cell monolayer integrity with time ( Figure 6), starting from 18 h of incubation, when the TEER value was about 80% of the level of the untreated cells. TEER values measured in monolayers treated with compounds 18e and 43a (20 μM), which did not significantly differ from control values throughout all the time points, showing no enhancement of cell monolayer permeability. Figure 6. Evaluation of cell monolayer permeabilization as transepithelial electrical resistance (TEER) assay. Caco-2 cell monolayers were incubated with LPS at 1 μg/mL (black squares) as negative control, compound 18e at 20 μM (blue triangles), compound 43a at 20 μM (green triangles), and Control (red circles) as positive control. Statistically significant differences are expressed as follows: * = p < 0.05 LPS/Control; *** = p < 0.001 LPS vs. Control. Each value represents the mean ± SD of independent experiments (n = 3).

Protective Effect of 18e on Vero-76 Cell from CVB5 Infection
Among the two interesting derivatives active against CVB5 and based on the described results, we selected compound 18e to verify whether it could hinder CVB5-induced apoptosis and preserve the monolayers viability. Vero-76 cells, growing in 12-well plates, were infected with CVB5 or left untreated. After adsorption, the cells were incubated in the absence or presence of 20 μM of compound 18e. The cells were incubated for 48 h and then stained with Annexin-V-fluorescein and propidium iodide and, subsequently, subjected to flow cytometry analysis. Figure 7 shows that CVB5 infection induced cell death mainly by apoptosis (27.05% ± 3.19 and 6.87 ± 0.29, early and late apoptotic cells, respectively), whereas in non-infected cells treated with 20 μM of compound 18e, a minimal number of early, late apoptotic and necrotic cells was detected, confirming the absence of cytotoxicity of the tested compound. In CVB5-infected cells, the administration of 18e at 20 μM concentration elicited a significant decrease in apoptotic cells (from 27.05 % ± 3.19 in untreated infected cells versus 6.35 % ± 1.47 in treated infected cells; p = 0.002). These results confirmed that CVB5 virus induces cell death by apoptosis and that compound 18e protects the cells from infection. Figure 6. Evaluation of cell monolayer permeabilization as transepithelial electrical resistance (TEER) assay. Caco-2 cell monolayers were incubated with LPS at 1 µg/mL (black squares) as negative control, compound 18e at 20 µM (blue triangles), compound 43a at 20 µM (green triangles), and Control (red circles) as positive control. Statistically significant differences are expressed as follows: * = p < 0.05 LPS/Control; *** = p < 0.001 LPS vs. Control. Each value represents the mean ± SD of independent experiments (n = 3).

Protective Effect of 18e on Vero-76 Cell from CVB5 Infection
Among the two interesting derivatives active against CVB5 and based on the described results, we selected compound 18e to verify whether it could hinder CVB5-induced apoptosis and preserve the monolayers viability. Vero-76 cells, growing in 12-well plates, were infected with CVB5 or left untreated. After adsorption, the cells were incubated in the absence or presence of 20 µM of compound 18e. The cells were incubated for 48 h and then stained with Annexin-V-fluorescein and propidium iodide and, subsequently, subjected to flow cytometry analysis. Figure 7 shows that CVB5 infection induced cell death mainly by apoptosis (27.05% ± 3.19 and 6.87 ± 0.29, early and late apoptotic cells, respectively), whereas in non-infected cells treated with 20 µM of compound 18e, a minimal number of early, late apoptotic and necrotic cells was detected, confirming the absence of cytotoxicity of the tested compound. In CVB5-infected cells, the administration of 18e at 20 µM concentration elicited a significant decrease in apoptotic cells (from 27.05% ± 3.19 in untreated infected cells versus 6.35% ± 1.47 in treated infected cells; p = 0.002). These results confirmed that CVB5 virus induces cell death by apoptosis and that compound 18e protects the cells from infection.

Virucidal Activity
To determine whether compound 18e acts directly on the viral particle leading to infectivity inactivation, a virucidal assay against CVB5 virions was conducted. As shown in Figure 8, any virucidal effect was detected testing 18e at the concentration of 20 μM at either 4 °C or 37 °C, since no difference between the titers of CVB5 treated at the two different temperatures was recorded. These results suggested that the inhibitions detected by the plaque reduction assay, reported in Table 1, may result from the interference with a CVB5 replication cycle stage.

Virucidal Activity
To determine whether compound 18e acts directly on the viral particle leading to infectivity inactivation, a virucidal assay against CVB5 virions was conducted. As shown in Figure 8, any virucidal effect was detected testing 18e at the concentration of 20 µM at either 4 • C or 37 • C, since no difference between the titers of CVB5 treated at the two different temperatures was recorded. These results suggested that the inhibitions detected by the plaque reduction assay, reported in Table 1, may result from the interference with a CVB5 replication cycle stage. In order to investigate the potential inhibitory mechanism for derivative 18e, a cell pre-treatment and a time course assay were performed on Vero-76 monolayers. For cell pre-treatment test, Vero cells were incubated with an active and not cytotoxic concentra-  In order to investigate the potential inhibitory mechanism for derivative 18e, a cell pre-treatment and a time course assay were performed on Vero-76 monolayers. For cell pre-treatment test, Vero cells were incubated with an active and not cytotoxic concentration of compound 18e (20 µM) for 2 h. The unbound compound was then removed, the cells infected with CVB5 for 2 h at room temperature and then washed, overlayed with new media and incubated to 37 • C for 3 days. The yield of viral particles was then determined by plaque assay and results are reported in Figure 9. Under the described experimental conditions, a decrease in viral load from 3 × 10 5 PFU/mL (not treated control) to 7× 10 4 (18e-treated) was detected.

Time of Addition (ToA)
In order to investigate the potential inhibitory mechanism for derivative 18e, a cell pre-treatment and a time course assay were performed on Vero-76 monolayers. For cell pre-treatment test, Vero cells were incubated with an active and not cytotoxic concentration of compound 18e (20 μM) for 2 h. The unbound compound was then removed, the cells infected with CVB5 for 2 h at room temperature and then washed, overlayed with new media and incubated to 37 °C for 3 days. The yield of viral particles was then determined by plaque assay and results are reported in Figure 9. Under the described experimental conditions, a decrease in viral load from 3 × 10 5 PFU/mL (not treated control) to 7× 10 4 (18e-treated) was detected. The antiviral effect was kept even in the time of addition assay when the compound 18e was added during the infection period. No titer reduction was observed adding 18e during subsequent steps of the replication cycle. These findings prompted us to The antiviral effect was kept even in the time of addition assay when the compound 18e was added during the infection period. No titer reduction was observed adding 18e during subsequent steps of the replication cycle. These findings prompted us to hypothesize a possible inhibition during an early phase of infection, by reducing the viral attachment process to the host cell.

Chemistry
Melting points were carried out with a Köfler hot stage or Digital Electrothermal melting point apparatus. Nuclear magnetic resonance ( 1 H NMR and 13 C NMR-APT) spectra were determined in CDCl 3 or DMSO-d 6 and were recorded with a Bruker Avance III 400 NanoBay. Chemical shifts are reported in parts per million (ppm) downfield from tetramethylsilane (TMS) used as the internal standard. Splitting patterns were designated as follows: s, singlet; d, doublet; t, triplet; q, quadruplet; quin, quintet; sext, sextet; sept, septet; m, multiplet; br s, broad singlet; dd, doublet of doublets. Mass spectra (MS) were performed on combined Liquid Chromatograph-Agilent 1100 series Mass Selective Detector (MSD). Analytical thin-layer chromatography (TLC) was performed on Merck silica gel F-254 plates. Pure compounds showed a single spot in TLC. For flash chromatography, Merck silica gel 60 was used with particle sizes 0.040 and 0.063 mm (230 and 400 mesh ASTM).

Cells and Viruses
Cell lines were purchased from American Type Culture Collection (ATCC). The absence of mycoplasma contamination was determined periodically by the Hoechst staining method. Cell lines supporting the multiplication of RNA and DNA viruses were the fol- Viruses were maintained in our laboratory and propagated in appropriate cell lines. All viruses were stored in small aliquots at −80 • C until use.

Cytotoxicity Assay
MDBK and BHK-21 cells were seeded at an initial density of 6 × 10 5 and 1 × 10 6 cells/mL, respectively, in 96-well plates containing minimum essential medium with Earle's salts (MEM-E), L-glutamine, 1 mM sodium pyruvate and 25 mg/L kanamycin, supplemented with 10% horse serum (MDBK) or 10% fetal bovine serum, FBS (BHK-21). Vero-76 cells were seeded at an initial density of 5 × 10 5 cells/mL in 96-well plates containing in Dulbecco's modified eagle medium (D-MEM) with L-glutamine and 25 mg/L kanamycin, supplemented with 10% FBS. Cell cultures were then incubated at 37 • C in a humidified, 5% CO 2 atmosphere, in the absence or presence of serial dilutions of test compounds. The test medium used for cytotoxic and antiviral assay contained 1% of the appropriate serum. Cell viability was determined after 72 or 96 h at 37 • C by the MTT method for MDBK, BHK-21 and Vero-76 cells [58].

Transepithelial Electrical Resistance (TEER) Assay
The cytotoxicity of the compounds 18e and 43a was tested on intestinal epithelial cell by estimating the TEER (Transepithelial Electrical Resistance) values as a measure of cell monolayer integrity. Caco-2 cells (ECACC Salisbury, Wiltshire UK) were cultured in Dulbecco's modified eagle's medium (DMEM), supplemented with 10% heat-inactivated bovine serum, 100 U/mL penicillin, 2 mM l-glutamine, 1% non-essential amino acids, and 100 mg/mL streptomycin at 37 • C in a humidified atmosphere of 5% CO 2 , replacing the medium twice a week [59]. All cell culture materials were purchased from Euroclone (Milan, Italy). Caco-2 cells (5 × 10 4 cells/well), at passage 31-40, were grown in 12 mm i.d. Transwell inserts (polycarbonate membrane, 0.4 µm pore size) (Corning Costar Corp., New York, NY, USA) and culture medium was dispensed both in the apical (0.5 mL) and in the basolateral (1.5 mL) compartment of each well. Resistance was assessed using Millicell-ERS voltohmmeter (Millicell-ERS system, Millipore, Bedford, MA, USA). After cell differentiation (>14 days), only cell monolayers in inserts with TEER values >300 Ω/cm 2 were considered for the experiment [60]. Then, the compounds 18e and 43a (final concentration 30 µM) and, as a proinflammatory agent, the Gram-negative endotoxin lipopolysaccharide (LPS, 100 µg/mL) were added in the culture medium and TEER values were measured at intervals of 3, 18, 24, 36, 48, 60, and 72 h and reported as percentage of the corresponding TEER value at time zero (T = 0).

Apoptosis Assay
To evaluate the levels of apoptosis following 18e derivative treatment, a flow cytometric analysis, using the cell apoptosis kit Annexin V/Propidium Iodide (PI) double staining uptake (Invitrogen, Life Technologies, Italy), was used. Vero-76 cells, at the density of 3 × 10 5 cells/mL, were seeded in 12-well plates (Corning, New York, NY, USA) with complete medium (described in the cell culture section). After CV-B5 viral adsorption, the cells were incubated in the absence or presence of different concentrations of 18e for 48 h, until the cytopathic effect CPE of the virus control reached 70-80%. Cells were then washed once with PBS 1 X and re-suspended in 100 µL of Annexin binding buffer plus 1 µL of Annexin V and 1 µL of PI. Then, the reaction was performed in the dark for 15 minutes at room temperature. Stained cells were then analyzed by flow cytometry, measuring the fluorescence emission at 530 and 620 nm using a 488 nm excitation laser (MoFloAstrios EQ, Beckman Coulter, Pasadena, CA). Cell apoptosis was analyzed using the software Summit Version 6.3.1.1, Beckman Coulter.

Antiviral Assay
Compound's activity against YFV and Reo-1 was based on inhibition of virus-induced cytopathogenicity in BHK-21 cells acutely infected with a m.o.i. of 0.01. Compound's activity against BVDV was based on inhibition of virus-induced cytopathogenicity in MDBK cells acutely infected with a m.o.i. of 0.01. After a 3-or 4-day incubation at 37 • C, cell viability was determined by the MTT method, as described by Pauwels et al. (1988). The compound's activity against CVB5, Sb-1, VSV, VV, RSV A2, and HSV-1 was determined by plaque reduction assays in infected cell monolayers, as described by Sanna et al. [61].

Virucidal Activity Assay
Benzotriazole derivatives (20 µM) were incubated with 1 × 10 5 PFU/mL of CVB5, at either 4 or 37 • C for 1 h. The mix without test sample was employed as a control. After incubation period, samples were serially diluted in media and titers were determined on Vero-76 cells CVB5 at high dilutions, at which the derivative was not active. Titers were then determined by plaque assay in Vero-76 cells.

Cell Pre-Treatment Assay
The monolayers of Vero-76 cell seeded in 24-well plates were incubated with 20 µM concentration of compound 18e for 2 h. After the removal of the test compound and two washes, the cells were infected with CVB5. After the adsorption of the virus to the cells, the inoculum was removed and the monolayers were overlaid with fresh medium, incubated for 3 days at 37 • C, and then virus titers were determined by plaque assay.

Time of Addition Assay
The monolayers of Vero-76 cells seed in 24-well tissue culture plates were infected for 1 h at room temperature with CVB5 dilutions to give final m.o.i. of 1. After adsorption, the monolayers were washed two times with D-MEM medium with L-glutamine, supplemented with 1% inactivated FBS, 1 mM sodium pyruvate and 0.025 g/L kanamycin (maintenance medium), and incubated with the same medium at 5% CO 2 and 37 • C (time zero). Vero-76 cells CVB5 were treated with benzotriazole derivative (20 µM) or reference for 1 h during infection period (at -1 to 0) and at specific time point, 0 to 2, 2 to 4, 4 to 6, post infection. After incubation period, the monolayers were washed two times with maintenance medium and incubated with fresh medium until 12 h post infection. Then, the plates were frozen at −80 • C and the viral titers were determined by plaque assay.

Statistical Analysis
All biological experiments were independently repeated at least three times. The data are reported as mean ± standard deviation (SD). The statistical significance (** p = 0.002) was performed in GraphPad Prism (San Diego, CA, USA.)

Experimental
The chemical characterization of the selected and deeply analyzed compounds 18e and 43a are reported below, all the other compounds' analysis can be found in the Supple-

Conclusions
In this work, we reported the synthesis and characterization of a large series of benzotriazole-based derivatives variously functionalized on the main core and equipped with an aromatic or aliphatic chain. Compounds were assayed against a wide panel of viruses and the obtained results allowed a SARs analysis to highlight the moieties eventually endowed with antiviral activity. Most of the active compounds showed a specific activity against CVB5. Notably, derivative 18e was found to be endowed with a considerable anti-enteroviral activity coupled with a cytotoxic profile in the high micromolar range and was selected to deepen its mechanism of action. TEER experiment was run on human epithelial cells and the results confirmed the safety of compound 18e. When analyzed in apoptotic assay, this derivative protected cells from the CVB5-induced apoptosis. In the following time course assay, our compound displayed its utmost activity during the pretreatment and infection period. So far, these findings prompted us to speculate on the main involvement of 18e during the entry process of the virus and suggested our benzotriazole derivative as a potential anti-CVB5 agent worth investigating and optimizing further.